Major depressive disorder (MDD) has been associated with abnormally reduced function of central serotonergic systems by various types of evidence. One instructive paradigm for investigating the relationship between serotonergic function and depression has involved the mood response to tryptophan depletion (TD), achieved by oral loading with all essential amino acids excepting the 5-HT precursor, tryptophan. We obtained preliminary evidence that the mood lowering effect of TD depends upon the genotype for a functional polymorphism in the promoter region of the 5-HT transporter (5-HTT), designated 5-HTTLPR, as well as upon family history. In healthy females the s-allele and a positive family history for mood disorders appeared to be additive risk factors for the development of depressive symptoms during TD. This year we reported that the glucose metabolic changes under TD not only differentiate healthy controls and subjects with MDD, but also can differentiate between s-carriers and l-homozygotes for the 5-HTTLPR polymorphism. These data added strong evidence to the literature that the s-allele is functionally relevant in the brain. This was important to elucidate, because the s-allele had been linked to an increased risk of developing depression within the context of stress. The current study employed quantitative PET imaging of cerebral blood flow (CBF) and glucose metabolism to investigate the effect of variant 5-HTTLPR genotypes on the neurophysiological response to TD. We also examined the relationship between 5-HTTLPR genotypes and the TD effect on PFC metabolic activity and whether reduction in PFC metabolism in response to TD would occur to a greater extent in subjects with the s/s allele and in subjects with a single s allele plus a family history of depression. We also examined whether this reduction in PFC metabolic activity is unique to subjects who develop depressive symptoms during TD. In addition, based upon evidence that 5-HT inhibits neuronal activity in the amygdala, and modulates transmission of emotionally-salient sensory information from the sensory cortices to the amygdala, we tested the hypothesis that in MDD, reduced serotonin function associated with TD may disinhibit the amygdaloid response to sensory stimulation. This hypothesis was explored by assessing the physiological responses of the amygdala to sensory stimuli that normally activate the amygdala, namely pictures of human faces that show fearful or sad emotional expressions. We were particularly interested in determining whether the amygdala CBF response to emotional stimuli during TD is most prominently increased in subjects carrying the s-allele of the 5-HTTLPR, and whether it is unique to subjects who develop depressive symptoms during TD. This year we reported the results of our investigation of the relationships between genotype, history of MDD, and serotonin depletion on the processing of emotionally valenced words. Recovered subjects with a history of MDD and healthy controls underwent functional MRI scanning while performing an "affective shift task" (in which subjects alternate attention between detecting sad versus happy words) in both the tryptophan depleted and the control condition. Thirty-six unmedicated-remitted subjects with MDD and 36 healthy controls were studied in previous years in a double-blind, placebo-controlled, randomized (according to 5-HTTLPR genotype) crossover study. The data analysis has continued and one manuscript has been published to describe the effects of genotype on the brain regions that change their activity during serotonin depletion and during depressive relapse. A second manuscript is in preparation to show that genotype (for the 5-HTTLPR genotype polymorphism) and phenotype (healthy control, MDD in depressive relapse, and MDD with maintained recovery) can be classified correctly in most cases by their metabolic response to tryptophan depletion. In addition, the cerebral blood flow (CBF) response to emotional faces was assessed in these cases and associated with a gene mutation for an adrenergic receptor that had been associated with increasing the risk for developing depression. This study also demonstrated abnormalities in the CBF responses to sad faces that were abnormal in recovered cases with MDD. In the fMRI study, 13 additional recovered patients with a history of MDD and 13 additional healthy controls werestudied and the results analyzed. A manuscript has published which describes the effects of tryptophan depletion on emotional processing in healthy humans, and another manuscript has been published which reports the differential effects of tryptophan depletion on both the behavioral and the neural responses to performing the task between the recovered MDD and control groups. This past year we extended this sample size and also examined the effects of a genetic polymorphism in the serotonin transporter on these data. We learned that this genetic variant, which has been associated with the vulnerability to depression and appears to have a functional effect on the serotonergic system, also affects the brain's responses to emotional words. In particular the subgenual anterior cingulate, a structure implicated in both the pathophysiology of depression and the mechanisms of antidepressant treatment, shows responses to sad words that differ on the basis of the polymorphism which particularly increases the vulnerability to depression.